Humanized IL-6 and IL-6 receptor

ABSTRACT

Mice that comprise a replacement of endogenous mouse IL-6 and/or IL-6 receptor genes are described, and methods for making and using the mice. Mice comprising a replacement at an endogenous IL-6Rα locus of mouse ectodomain-encoding sequence with human ectodomain-encoding sequence is provided. Mice comprising a human IL-6 gene under control of mouse IL-6 regulatory elements is also provided, including mice that have a replacement of mouse IL-6-encoding sequence with human IL-6-encoding sequence at an endogenous mouse IL-6 locus.

This application is a continuation of U.S. patent application Ser. No.13/662,880, filed Oct. 29, 2012, now U.S. Pat. No. 8,878,001, whichclaims the benefit under 35 USC §119(e) of U.S. Provisional PatentApplication Ser. No. 61/552,900, filed on Oct. 28, 2011, and U.S.Provisional Patent Application Ser. No. 61/556,579, filed on Nov. 7,2011; each provisional application is hereby incorporated by reference.

FIELD OF INVENTION

Non-human animals having a replacement of the endogenous non-humananimal IL-6 and/or IL-6 receptor genes are provided. IL-6 and/or IL-6receptor genes of the non-human animal are replaced, at the endogenousnon-human loci, with human IL-6 and/or humanized IL-6 receptor genescomprising human sequence. Non-human animals that have human IL-6 and/orhumanized IL-6 receptor genes, wherein the non-human animals do notexhibit one or more pathologies that are characteristic of non-humananimals transgenic for human IL-6.

BACKGROUND

Mice transgenic for a human IL-6 gene are known in the art. However,random insertion of a human IL-6 transgene into the mouse genome resultsin poorly regulated expression of the human IL-6 protein, whichmanifests itself in a variety of pathologies in such transgenic mice,including, but not limited to, plasmacytosis and glomerulonephritis. Asa result, these mice have limited usefulness.

There is a need for non-human animals, e.g., mice and rats, the expresshuman or humanized IL-6 and/or human or humanized IL-6 receptor. Thereis a need for such humanized mice that do not exhibit one or morepathologies exhibited by transgenic hIL-6 mice.

SUMMARY

In one aspect, genetically modified non-human animals are provided thatcomprise a replacement at an endogenous IL-6 and/or IL-6 receptor locusof a gene encoding an endogenous IL-6 and/or IL-6 receptor with a geneencoding a human or humanized IL-6 and/or IL-6 receptor. Murine animalsare provided that comprise a replacement of an endogenous IL-6 gene, atan endogenous murine IL-6 locus, with a human IL-6 gene; and/or thatcomprise a replacement of an endogenous IL-6 receptor gene (ornucleotide sequence encoding an ectodomain thereof) with a human IL-6receptor gene (or nucleotide sequence encoding an ectodomain thereof).

In one aspect, genetically modified murine animals are provided thatexpress a human IL-6 gene under the control of endogenous murinepromoter and/or endogenous murine regulatory elements, from anendogenous murine IL-6 locus.

In one aspect, genetically modified murine animals are provided thatexpress a human IL-6 receptor gene (or a gene encoding a humanectodomain and mouse transmembrane and intracellular domains) under thecontrol of endogenous murine promoter and/or endogenous murineregulatory elements, from an endogenous murine IL-6 receptor locus.

In one aspect, a genetically modified animal (e.g., a murine animal,e.g., a mouse or rat) is provided that expresses a human IL-6 protein,wherein the non-human animal does not exhibit a pathology selected fromplasmacytosis, glomerulonephritis, glomerulosclerosis,mesangio-proliferative glomerulonephritis, intestinal lymphoma, kidneylymphoma, splenomegaly, lymph node enlargement, liver enlargement,megakaryocytes in bone marrow, compacted abnormal plasma cells,infiltration of plasma cells into lung or liver or kidney, mesangialcell proliferation in kidney, cerebral overexpression of IL-6, ramifiedmicroglial cells in white matter, reactive astrocytosis in brain, kidneyfailure, elevated megakaryocytes in spleen, muscle wasting (e.g.,gastrocnemius muscle wasting), elevated muscle cathepsins B and B+L(e.g., around 20-fold and 6-fold), and a combination thereof.

In one embodiment, the non-human animal comprises a normal B cellpopulation. In one embodiment, the normal B cell population isapproximately the same in number and immunophenotype as a wild-typeanimal, e.g., a wild-type mouse.

In one embodiment, the non-human animal is murine (e.g., a mouse or rat)and expresses human IL-6 (hIL-6) in serum at a level below about 800pg/mL, below about 700, 600, 500, 400, 300, or 200 pg/mL. In a specificembodiment, the murine animal expresses hIL-6 in serum at a level ofabout 50 to about no more than 200 pg/mL, in another embodiment about75-125 pg/mL, in another embodiment at about 100 pg/mL.

In one aspect, a non-human animal is provided that expresses hIL-6and/or hIL-6R, wherein the non-human animal expresses hIL-6 and/orhIL-6R from an endogenous non-human IL-6 locus and/or an endogenousnon-human hIL-6R locus. In a specific embodiment, the non-human animalis murine (e.g., mouse or rat).

In one aspect, a genetically modified mouse is provided that expresseshIL-6 from an endogenous mouse IL-6 locus, wherein the endogenous mouseIL-6 gene has been replaced with a hIL-6 gene.

In one embodiment, the mouse comprises a cell that expresses an IL-6receptor (IL-6R) that comprises a human ectodomain on the surface of thecell. In one embodiment, the cell is a lymphocyte. In one embodiment,the lymphocyte is a B cell.

In one embodiment, about 6.8 kb at the endogenous mouse IL-6 locus,including exons 1 through 5 and a 3′ untranslated sequence, is deletedand replaced with about 4.8 kb of human IL-6 gene sequence comprisingexons 1 through 5 of the human IL-6 gene. In a specific embodiment, thehuman IL-6 gene comprises exons 1 through 5 of the human IL-6 gene ofhuman BAC CTD-2369M23.

In one aspect, a genetically modified mouse is provided that expressesIL-6 from a human IL-6 gene, wherein the mouse expresses human IL-6 inits serum.

In one embodiment, the mouse serum exhibits a serum concentration ofhuman IL-6 of about 25 to about 300 pg/mL, 50 to about 250 pg/mL, 75 toabout 200 pg/mL, or 100 to about 150 pg/mL. In a specific embodiment,the level of human IL-6 in the serum of the mouse is about 100 pg/mL.

In one embodiment, the level of a pan B cell-specific marker in bonemarrow of the mouse is about the same as that of a wild-type mouse. Inone embodiment, the level of a pan B cell-specific marker in spleen isabout the same as that of a wild-type mouse. In one embodiment, the panB cell-specific marker is selected from B220, CD19, CD20, CD22, CD79a,CD79b, L26, and Pax-5 (BSAP).

In one aspect, a genetically modified mouse is provided that expresseshIL6, wherein the mouse does not exhibit a feature selected fromplasmacytosis, splenomegaly, lymph node enlargement, compacted abnormalplasma cells, and a combination thereof.

In one embodiment, the mouse comprises a spleen that is about the sameweight (per body weight) as a wild-type mouse. In one embodiment, thelymph nodes of the mouse are about the same weight (per body weight) asa wild-type mouse. In one embodiment, plasma cells of the mouse do notexhibit plasmocytosis characteristic of mice that overexpress humanIL-6.

In one embodiment, the mouse does not exhibit glomerulonephritis.

In one embodiment, the mouse exhibits a mesangial cell level comparableto a wild-type mouse.

In one aspect, a genetically modified mouse is provided that expresseshIL6 from an endogenous mouse IL-6 locus, wherein the endogenous mouseIL-6 gene has been replaced with a hIL-6 gene, wherein the mouse doesnot exhibit a feature selected from a morphologically detectableneuropathology, a reactive astrocytosis, and a combination thereof. Inone embodiment, the mouse comprises a brain that is morphologicallyindistinct from a wild-type mouse brain. In one embodiment, the mousecomprises brain tissue that exhibits a level of reactive astrocytosisthat is no higher than that of a wild-type mouse.

In one embodiment, the mouse does not express human IL-6 in neurons. Inone embodiment, the mouse comprises activated astrocyte levels that arecomparable to activated astrocyte levels in a wild-type mouse.

In one embodiment, the mouse comprises ramified microglial cells in itswhite matter, wherein the ramified microglial cells are present in anamount equivalent to an amount of ramified microglial cells in awild-type mouse.

In one embodiment, the mouse does not exhibit a reactive atrocytosis. Inone embodiment, the white matter of the mouse is morphologicallyindistinct from the white matter of a wild-type mouse. In oneembodiment, the white matter of the mouse is histologically indistinctfrom a wild-type mouse white matter with respect to histochemicalstaining of reactive astrocytes.

In one embodiment, the mouse comprises a brain that is morphologicallyindistinct from a wild-type mouse brain. In one embodiment, the mousecomprises brain tissue that exhibits a level of reactive astrocytosisthat is no higher than that of a wild-type mouse.

In one aspect, a genetically modified mouse is provided that expresseshIL6 from an endogenous mouse IL-6 locus, wherein the endogenous mouseIL-6 gene has been replaced with a hIL-6 gene, wherein the mouse doesnot exhibit a feature selected from a life span shortened by about 50%or more, kidney failure, hypergammaglobulinemia, elevated megakaryocytesin spleen, elevated megakaryocytes in bone marrow, plasmacytosis ofspleen, plasmacytosis of thymus, plasmacytosis of lymph nodes,glomerulonephritis, glomerulosclerosis, and a combination thereof.

In one embodiment, the mice have a life span that exceeds 20 weeks. Inone embodiment, the mice have a life span that exceeds 30 weeks, 40weeks, or 50 weeks. In one embodiment, the mice exhibit a life spanabout equal to that of a wild-type mouse of the same strain.

In one embodiment, the mice exhibit a level of megakaryocytes in spleenthat is no more than about the splenic megakaryocyte level of awild-type mouse

In one embodiment, the mice comprise lymphoid organs that areessentially devoid of abnormal and compactly arranged plasmacytoidcells.

In one embodiment, the mice exhibit gamma globulin serum levelsequivalent to gamma globulin serum levels in wild-type mice. In oneembodiment, the levels of α1- and β-globulin in serum of the mice areequivalent to α1- and β-globulin serum levels of wild-type mice of thesame strain.

In one aspect, a genetically modified mouse is provided that expresseshuman IL-6 from an endogenous mouse IL-6 locus, wherein the endogenousmouse IL-6 gene has been replaced with a hIL-6 gene, wherein the mousedoes not exhibit a feature selected from muscle wasting, an elevatedcathepsin B level as compared with a wild-type mouse of the same strain,an elevated cathepsin A+B level as compared with a wild-type mouse ofthe same strain, an increased liver weight as compared with a wild-typemouse of the same strain, and a combination thereof.

In one embodiment, the weight of the liver of the mouse is about 800-900mg at 12 weeks.

In one embodiment, the mouse exhibits a cathepsin B level throughout itslife span that is no more than about the level observed in a wild-typemouse. In one embodiment, the mouse exhibits a cathepsin A+B levelthroughout its life span that is no more than about the level observedin a wild-type mouse.

In one embodiment, the mouse as an adult exhibits a gastrocnemus muscleweight that is within about 10% of the weight of a wild-type mouse ofthe same strain. In one embodiment, the mouse as an adult exhibits agastrocnemus muscle weight that is about the same as that of a wild-typemouse.

In one aspect a mouse is provided that comprises a nucleotide sequenceencoding a human IL-6 protein, wherein the nucleotide sequence encodingthe human IL-6 protein replaces in whole or in part an endogenousnucleotide sequence encoding and endogenous mouse IL-6 protein.

In one aspect, a mouse is provided that comprises a replacement at anendogenous mouse IL-6 receptor locus of mouse IL-6Rα ectodomain with anectodomain sequence of a human IL-6Rα to form a chimeric human/mouseIL-6Rα gene.

In one embodiment, the chimeric IL-6Rα gene is under the control of amouse promoter and/or mouse regulatory elements at the endogenous mouseIL-6Rα locus.

In one embodiment, about 35.4 kb of mouse IL-6Rα ectodomain-encodingsequence is replaced with about 45.5 kb of human IL-6Rectodomain-encoding sequence.

In one embodiment, the human IL-6R ectodomain-encoding sequenceencompasses the first (ATG) codon in exon 1 through exon 8.

In one embodiment, the mouse IL-6Rα sequence that is replaced includes acontiguous sequence that encompasses exons 1 through 8. In a specificembodiment, exons 1 through 8 and a portion of intron 8 is deleted.

In one aspect, a genetically modified mouse is provided, comprising areplacement at an endogenous mouse IL-6 locus of a mouse gene encodingIL-6 with a human gene encoding human IL-6, wherein the human geneencoding human IL-6 is under control of endogenous mouse regulatoryelements at the endogenous mouse IL-6 locus.

In one embodiment, the human gene encoding human IL-6 is a human IL-6gene of BAC ID CTD-2369M23.

In one embodiment, the mouse expresses a mouse IL-6Rα. In oneembodiment, the mouse expresses a human IL-6Rα. In one embodiment, thehumanized IL-6Rα comprises a human ectodomain. In one embodiment, thehumanized IL-6Rα comprises a mouse transmembrane domain and a mousecytoplasmic domain. In one embodiment, the mouse expresses a humanizedIL-6Rα that comprises a humanization of ectodomain but not transmembraneand/or cytosolic domain.

In one embodiment, the mouse does not exhibit a feature selected fromplasmocytosis, glomerulosclerosis, glomerulonephritis, kidney failure,hypergammaglobulinemia, elevated megakaryocytes in spleen, elevatedmegakaryocytes in bone marrow, splenomegaly, lymph node enlargement,compacted abnormal plasma cells, and a combination thereof.

In one aspect, a genetically modified mouse is provided, comprising ahumanization of an endogenous mouse IL-6Rα gene, wherein thehumanization comprises a replacement of mouse IL-6Rα ectodomain-encodingsequence with human IL-6Rα ectodomain-encoding sequence at theendogenous mouse IL-6Rα locus.

In one embodiment, a contiguous mouse sequence comprising mouse exons 1through 8 is replaced with a contiguous genomic fragment of human IL-6Rαsequence encoding a human IL-6Rα ectodomain. In one embodiment, thecontiguous genomic fragment of human IL-6Rα sequence encoding theectodomain is from BAC CTD-2192J23.

In one embodiment, the mouse further comprises a humanized IL-6 gene. Inone embodiment, the mouse comprises a replacement at an endogenous mouseIL-6 locus of a mouse IL-6 gene with a human IL-6 gene. In oneembodiment, the humanized IL-6 gene is under control of endogenous mouseregulatory elements.

In one aspect, a method is provided for making a humanized mouse,comprising replacing a mouse gene sequence encoding mouse IL-6 with ahuman gene encoding human IL-6.

In one embodiment, the replacement is at an endogenous mouse IL-6 locus,and the human gene encoding human IL-6 is operably linked to endogenousmouse regulatory sequences.

In one aspect, a method for making a humanized mouse is provided,comprising replacing mouse exons encoding ectodomain sequences of mouseIL-6Rα with a human genomic fragment encoding ectodomain sequences ofhuman IL-6Rα to form a humanized IL-6Rα gene.

In one embodiment, the replacement is at an endogenous mouse IL-6Rαlocus, and the humanized IL-6Rα gene is operably linked to endogenousmouse regulatory sequences.

In one aspect, a genetically modified mouse is provided, comprising ahumanized IL-6Rα gene comprising a replacement of mouseectodomain-encoding sequence with human ectodomain sequence, wherein thehumanized IL-6Rα gene comprises a mouse transmembrane sequence and amouse cytoplasmic sequence; wherein the mouse further comprises a geneencoding a human IL-6, wherein the gene encoding a human IL-6 is undercontrol of endogenous mouse IL-6 regulatory elements.

In one embodiment, the mouse is incapable of expressing a fully mouseIL-6Rα and incapable of expressing a mouse IL-6.

In various aspects, the genetically modified mice described hereincomprise the genetic modifications in their germline.

In one aspect, a tissue, cell, or membrane fragment from a mouse asdescribed herein is provided.

In one embodiment, the tissue or cell is from a mouse that expresses ahuman IL-6 protein, but that does not express a mouse IL-6 protein. Inone embodiment, the tissue or cell is from a mouse that expresses ahumanized IL-6Rα protein, but not a mouse IL-6Rα protein. In oneembodiment, the humanized IL-6Rα protein comprises a human ectodomainand a mouse transmembrane domain and a mouse cytosolic domain. In oneembodiment, the tissue or cell is from a mouse that expresses a humanIL-6, a humanized IL-6Rα, and that does not express a mouse IL-6 anddoes not express an IL-6Rα that comprises a mouse ectodomain.

In one aspect, an ex vivo complex of a mouse cell bearing a humanizedIL-6Rα (human ectodomain and mouse transmembrane and mouse cytoplasmicdomain) and a human IL-6 is provided.

In one aspect, a mouse embryo comprising a genetic modification asdescribed herein is provided.

In one aspect, a mouse host embryo is provided that comprises a donorcell that comprises a genetic modification as described herein.

In one aspect, a pluripotent or totipotent non-human animal cellcomprising a genetic modification as described herein is provided. Inone embodiment, the cell is a murine cell. In one embodiment, the cellis an ES cell.

In one aspect, a mouse egg is provided, wherein the mouse egg comprisesan ectopic mouse chromosome, wherein the ectopic mouse chromosomecomprises a genetic modification as described herein.

In one aspect, the mouse, embryo, egg, or cell that is geneticallymodified to comprise a human IL-6 gene or human or humanized IL-6Rα geneis of a mouse that is of a C57BL strain selected from C57BL/A, C57BL/An,C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ,C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/OIa. In anotherembodiment, the mouse is a 129 strain selected from the group consistingof a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV,129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac),129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revisednomenclature for strain 129 mice, Mammalian Genome 10:836, see also,Auerbach et al (2000) Establishment and Chimera Analysis of 129/SyEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.In one embodiment, the mouse is Swiss or Swiss Webster mouse.

Each of the aspects and embodiments described herein are capable ofbeing used together, unless excluded either explicitly or clearly fromthe context of the embodiment or aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an illustration, not to scale, of the human (A) andmouse (B) IL-6 genomic loci. Exons I, II, III, IV, and V (in both humanand mouse) are indicated by closed boxes to the right in the figure.Selected putative regulatory regions are indicated by open boxes to theleft in the figure.

FIG. 2 shows acute phase response (mSAA level) in the presence orabsence of turpentine in wild-type mice, humanized ectodomain IL-6Rmice, and mice with humanized IL-6 and IL-6R genes.

FIG. 3 shows turpentine-dependent acute phase response (SAA) inwild-type mice the absence or presence of anti-mouse IL-6R antibody (A);and turpentine-dependent acute phase response in humanized IL-6/IL-6Rmice in the absence or present of anti-human IL-6R antibody (B).

FIG. 4 shows FACS analysis for splenic B cells of wild-type (A) andhumanized (B) IL-6 mice; pan B cell marker.

FIG. 5 shows FACS analysis for splenic T cells of wild-type (A) andhumanized (B) IL-6 mice; T helper cells and cytotoxic T cells.

FIG. 6 shows FACS analysis for splenic cells of wild-type (A) andhumanized (B) IL-6 mice; Ly6G/C(Gr1).

FIG. 7 shows FACS analysis for splenic cells of wild-type (A, C) andhumanized (B, D) IL-6 mice; NK cells (A-B) and granulocytes(Ly6G^(hi+)/CD11b^(hi+)) (C-D).

FIG. 8 shows FACS analysis for blood B cells of wild-type (A) andhumanized (B) IL-6 mice; pan B cell marker.

FIG. 9 shows FACS analysis for blood T cells of wild-type (A) andhumanized (B) IL-6 mice; T helper cells and cytotoxic T cells.

FIG. 10 shows FACS analysis for blood myeloid cells of wild-type (A) andhumanized (B) IL-6 mice; Gr1⁺ cells.

FIG. 11 shows FACS analysis for blood myeloid cells of wild-type (A) andhumanized (B) IL-6 mice; CD11b vs. Ly6G/C(Gr1).

FIG. 12 shows FACS analysis for blood myeloid cells of wild-type (A-B)and humanized (C-D) IL-6 mice; DX5 vs CD11b cells.

FIG. 13 shows FACS analysis of bone marrow IgM/CD24/B220 for wild-typeand humanized IL-6 mice. A: normal progression in bone marrow. B-D: FACSanalysis for wild-type (B), hIL-6 heterozygotes (C), and hIL-6homozygotes (D) (IgM staining).

FIG. 14 shows FACS analysis of bone marrow IgM/CD24/B220 for wild-typeand humanized IL-6 mice. A: normal progression in bone marrow. B-D: FACSanalysis for wild-type (B), hIL-6 heterozygotes (C), and hIL-6homozygotes (D) (CD24 staining).

FIG. 15 shows FACS analysis of bone marrow CD43 and B220 for wild-typeand humanized IL-6 mice. A: normal progression in bone marrow. B-D: FACSanalysis for wild-type (B), hIL-6 heterozygotes (C), and hIL-6homozygotes (D) (CD43 staining).

DETAILED DESCRIPTION

IL-6 and IL-6R

The IL-6 receptor (IL-6R) was long characterized as a receptor for a Bcell stimulatory factor (BSF-2, or B cell Stimulatory Factor 2; also,BCDF, or B Cell Differentiation Factor) responsible for inducing B cellsto synthesize immunoglobulin (Yamasaki et al. (1988) Cloning andExpression of the Human Interleukin-6(BSF-2/IFNβ 2) Receptor, Science241:825-828). IL-6 was first described as interferon-β2 as the result ofits discovery during a search for a virally-induced protein termedinterfereon-β, by treating human fibroblasts with dsRNA poly(I)poly(C)to induce an anti-viral response (Weissenbach et al. (1980) Twointerferon mRNAs in human fibroblasts: In vitro translation andEscherichia coli cloning studies, Proc. Natl Acad. Sci. USA77(12):7152-7156; Keller et al. (1996) Molecular and Cellular Biology ofInterleukin-6 and Its Receptor, Frontiers in Bioscience 1:d340-357).

The human cDNA encodes a 468 amino acid protein having a 19-mer signalsequence and a cytoplasmic domain of about 82 amino acids that lacks atyrosine kinase domain (see, Id.). The N-terminal (ectodomain) of theprotein has an Ig superfamily domain of about 90 amino acids, a250-amino acid domain between the Ig superfamily domain and themembrane, a transmembrane span of about 28 amino acids (see, Id.). Theectodomain of the receptor binds its ligand IL-6, which triggersassociation with gp130 in the membrane and it is this complex thatconducts signal transduction; the cytoplasmic domain reportedly does nottransduce signal (Taga et al. (1989) Interleukin-6 Triggers theAssociation of Its Receptor with a Possible Signal Transducer, gp130,Cell 58:573-581). Indeed, a soluble form of IL-6R lacking a cytoplasmicdomain can associate with IL-6 and bind gp130 on the surface of a celland effectively transduce signal (Id.).

The homology of hIL-6R and mIL-6R at the protein level is only about54%; the transmembrane domain has a homology of about 79%, whereas thecytoplasmic domain has a homology of about 54% (Sugito et al. (1990)).

The natural ligand for the IL-6R, IL-6, was first isolated from culturesof HTLV-1-transformed T cells (see, Hirano et al. (1985) Purification tohomogeneity and characterization of human B cell differentiation factor(BCDF or BSFp-2), Proc. Natl. Acad. Sci. USA 82:5490-5494). A human cDNAfor the IL-6 gene was cloned at least twice, once as BSF-2 (see, Hiranoet al. (1086) Complementary DNA fro a novel human interleukin (BSF-2)that induces B lymphocytes to produce immunoglobulin, Nature 324:73-76)and once as IFNβ 2 (see, Zilberstein et al. (1986) Structure andexpression of cDNA and genes for human interferon-beta-2, a distinctspecies inducible by growth-stimulatory cytokines, EMBO 5:2529-2537),although it has since been demonstrated that recombinant human IL-6exhibits no detectable IFN activity.

Human IL-6 is a 184-amino acid protein that exhibits only about 42%homology with mouse IL-6, although the genomic organization of the humanand mouse genes are basically the same, and the promoter regions of thehuman and mouse genes share a 400-bp stretch that is highly conserved(see, Tanabe et al. (1988) Genomic Structure of the Murine IL-6 Gene:High Degree Conservation of Potential Regulatory Sequences between Mouseand Human, J. Immunol. 141(11):3875-3881).

The human IL-6 gene is about 5 kb (Yasukawa et al. (1987) Structure andexpression of human B cell stimulatory factor-2 (BSC-2/IL-6) gene, EMBOJ. 6(10):2939-2945), whereas the mouse IL-6 gene is about 7 kb (Tanabeet al. (1988) Genomic Structure of the Murine IL-6 Gene: High DegreeConservation of Potential Regulatory Sequences between Mouse and Human,J. Immunol. 141(11):3875-3881). The mouse and human IL-6 genesreportedly share highly conserved 5′-flanking sequence important toregulation. A schematic diagram of the human and mouse IL-6 genomic lociis shown in FIG. 1 (not to scale). Exons I, II, III, IV, and V (in bothhuman and mouse) are indicated by closed boxes to the right in thefigure. Selected putative regulatory regions are indicated by open boxesto the left in the figure. The putative regulatory regions for humansare, from left to right, a glucocorticoid element from −557 to −552; anIFN enhancer core sequence from −472 to −468; a glucocorticoid elementfrom −466 to −461; an AT-rich region from −395 to −334, a consensus AP-1binding site from −383 to −277; an IFN enhancer core sequence from −253to −248; a GGAAA-containing motif from −205 to −192; a c-fos SREhomology sequence from −169 to −82 containing an IFN enhancer coresequence, a cAMP-response element, a GGAAA motif, a CCAAT box, and aGC-rich region; and AP-1 binding site from −61 to −55; and a CCAAT boxfrom −34 to −30. The putative regulatory regions for mouse are, fromleft to right, a GC rich region from −553 to −536, a glucocorticoidelement from −521 to −516 and from −500 to −495; a Z-DNA stretch from−447 to −396; an AP-1 binding site overlapping an IFN enhancer coresequence from −277 to −288, a GGAAA motif overlapping an IFN enhancercore sequence from −210 to −195; a c-fos SRE homology region from −171to −82 containing a cAMP response element, a GGAAA motif overlapping anIFN enhancer core sequence, and a GC-rich region; and, an AP-1 bindingsite from −61 to −55. Mouse codons I-V have lengths 19, 185, 114, 150,and 165, respectively. Mouse intron lengths are: I-II, 162 bp; II-III,1253 bp; III-IV, 2981 bp; IV-V, 1281 bp. Human codons I-V have lengths19, 191, 114, 147, and 165. Human intron lengths are I-II, 154; II-III,1047; III-IV, 706; IV-V, 1737. Genomic organization data are from Tanabeet al. (1988), and Yasukawa et al. (1987) Structure and expression ofhuman B cell stimulatory factor-2 (BSF-2/IL-6) gene, EMBO J.9(10):2939-2945.

It might be reasonable to presume that the mouse and human IL-6 genesappear to be similarly regulated based on the similarity of their5′-flanking sequence. A variety cell types exhibit enhanced IL-6expression in response to IL-1, TNF, PDGF, IFNβ, serum, poly(I)poly(C),and cycloheximide (see, Tanabe et al. (1988). IL-6 in humans mediatesthe acute phase response, hematopoiesis, B cell differentiation, T cellactivation, growth and/or differentiation and/or activation of a varietyof cell types (e.g., hepatocytes, fibroblasts, endothelial cells,neurons, pituitary cells, lymphomas, myelomas, breast carcinomas, NKcells, macrophages, osteoclasts, etc.) (reviewed in, e.g., Heinrich etal. (1990), Kishimoto et al. (1989), and Keller et al. (1996); Sugita etal. (1990) Functional Murine Interleukin Receptor with Intracisternal AParticle Gene Product at its Cytoplasmic Domain, J. Exp. Med.171:2001-2009).

In practice, however, mice transgenic for human IL-6 exhibit a panoplyof substantial and debilitating pathologies, reflecting a significantpleiotropy of the IL-6 gene. Transgenic mice comprising a 6.6-kbfragment containing the human IL-6 gene and a μ enhancer (Eμ) producehigh concentrations of hIL-6 and extremely high IgG1 levels (120- to400-fold over wild-type mice), reflecting an IL-6 deregulation that isaccompanied by plasmacytosis, mesangio-proliferative glomerulonephritis,and high bone marrow megakaryocyte levels (Suematsu et al. (1989) IgG1plasmacytosis in interleukin 6 transgenic mice, Proc. Natl Acad. Sci.USA 86:7547-7551). Aberrant regulation of IL-6 and/or IL-6R isassociated with myelomas, plastocytomas, rheumatoid arthritis,Castleman's disease, mesangial proliferative glomerulonephritis, cardiacmyxoma, plams cell neoplasias, psoriasis, and other disorders (see,Kishimoto, T. (1989) The Biology of Interleukin-6, Blood 74(1):1-10;Sugita et al. (1990); also, Hirano et al. (1990) Biological and clinicalaspects of interleukin 6, Immunology Today 11(12):443-449)). IL-6 isalso implicated in sustaining levels of intra-prostatic androgens duringandrogen deprivation treatment of prostate cancer patients by aparacrine and/or autocrine mechanism, potentially providingcastration-resistant prostate tumor growth (Chun et al. (2009)Interleukin-6 Regulates Androgen Synthesis in Prostate Cancer Cells,Clin. Cancer Res. 15:4815-4822).

The human protein is encoded as a 212 amino acid protein, in mature forma 184 amino acid protein following cleavage of a 28 amino acid signalsequence. It contains two N-glycosylation and two O-glycosylation sites,and human IL-6 is phosphorylated in some cells. The mouse protein isencoded as a 211 amino acid protein, in mature form a 187 amino acidprotein following cleavage of a 23 amino acid signal sequence.O-glycosylation sites are present, but not N-glycosylation sites. (Seereviews on IL-6, e.g., Heinrich et al. (1990) Interleukin-6 and theacute phase response, Biochem. J. 265:621-636.)

IL-6 function is pleiotropic. The IL-6 receptor is found on activated Bcells but reportedly not on resting B cells. In contrast, IL-6R is foundon resting T cells and can reportedly promote T cell differentiation,activation, and proliferation, including the differentiation of T cellsinto cytotoxic T lymphocytes in the presence of IL-2.

Humanized IL-6/IL-6R Ectodomain Mice and IL-6-Mediated Acute PhaseResponse

In humans, IL-6 induces the acute phase response. Early studies withhuman hepatocytes established that IL-6 induces acute phase proteinssuch as, e.g., C-reactive protein (CRP) and serum amyloid A (SAA) in adose-dependent and time-dependent manner (reviewed in Heinrich et al.(1990) Interleukin-6 and the acute phase response, Biochem. J.265:621-636). Non-human animals, e.g., mice or rats, comprisinghumanized IL-6 and IL-6R genes are therefore useful systems formeasuring the acute phase response mediated by human IL-6. Such animalsare also useful for determining whether a substance induces anIL-6-mediated acute phase response, by exposing a humanized IL-6/IL-6Ranimal as described herein to the substance, and measuring a level ofone or more acute phase response proteins (or RNAs). In one embodiment,the humanized animal is exposed to the substance in the presence of anantagonist of a human IL-6R, and a level of one or more acute phaseresponse proteins (or RNAs) is measured, wherein a reduction in a levelof an acute phase response protein (or RNA) in the presence of the humanIL-6R antagonist indicates a human IL-6R-mediated acute phase response.

Human IL-6 can bind both human IL-6R and mouse IL-6R; mouse IL-6 bindsmouse IL-6R but not human IL-6R (no binding of mIL-6 to hIL-6Rdetectable, whereas hIL-6 can compete with mIL-6 for binding mIL-6R;Coulie et al. (1989) High- and low-affinity receptors for murineinterleukin 6. Distinct distribution on B and T cells, Eur. J. Immunol.19:2107-211); see also, e.g., Peters et al. (1996) The Function of theSoluble Interleukin 6 (IL-6) Receptor In Vivo: Sensitization of HumanSoluble IL-6 Receptor Transgenic Mice Towards IL-6 and Prolongation ofthe Plasma Half-life of IL-6, J. Exp. Med. 183:1399-1406). Thus, humancells that bear hIL-6R in a mouse (e.g., in a xenogenic transplant)cannot rely on endogenous mIL-6 to carry out IL-6-mediated functions,including but not limited to the role of IL-6 blood cell or lymphocytedevelopment (e.g., hematopoiesis, B cell activation, T cell activation,etc.).

In a mixed in vivo system comprising a wild-type mouse IL-6 gene and ahuman IL-6R gene (but no mouse IL-6R gene), an acute phase responseinducer is not expected to induce detectable levels of acute phaseproteins that would indicate an acute phase response. However, ahumanized mouse as described herein, comprising a humanized IL-6 geneand an IL-6R gene comprising a humanized ectodomain sequence willrespond to an acute phase response inducer and exhibit acute phaseresponse proteins in serum. Mice wild-type for IL-6/IL-6R tested foracute phase proteins in the presence or absence of the acute phaseinducer turpentine showed a turpentine-dependent increase in acute phaseproteins. Mice with humanized IL-6 gene, but not IL-6R, showed no acutephase response in the presence of turpentine. But mice bearing both ahuman IL-6 gene and an IL-6R gene with a humanized ectodomain exhibiteda strong acute phase response (FIG. 2). The IL-6-mediated acute phaseresponse was IL-6 dependent in both wild-type mice (FIG. 3, top) and inhumanized IL-6/IL-6R ectodomain mice (FIG. 3, bottom), as evidenced bythe ability of the appropriate anti-IL-6R antibody to abrogate the acutephase response at a sufficiently high antibody dose. Thus, a doublehumanization of IL-6 and IL-6R recapitulates the wild-type IL-6-mediatedacute phase response with respect to serum acute phase proteins.

Genetically Modified Mice

Genetically modified mice are provided that express a human IL-6 and/ora humanized IL-6 receptor from endogenous mouse loci, wherein theendogenous mouse IL-6 gene and/or the endogenous mouse IL-6 receptorgene have been replaced with a human IL-6 gene and/or a human sequencecomprising a sequence that encodes an ectodomain of a human IL-6receptor. The genetically modified mice express the human IL-6 and/orhumanized IL-6 receptor from humanized endogenous loci that are undercontrol of mouse promoters and/or mouse regulatory elements. Thereplacement(s) at the endogenous mouse loci provide non-human animalsthat express human IL-6 and a humanized IL-6 receptor in a manner thatdoes not result in the panoply of substantial pathologies observed inIL-6 transgenic mice known in the art.

Transgenic mice that express human IL-6 are known in the art. However,they generally suffer from significant pathologies that severely limittheir usefulness. Humanized mice as described herein express a humanIL-6 and/or humanized IL-6 receptor under the control of endogenousmouse regulatory elements at endogenous mouse IL-6 and IL-6Rα loci.These mice, in contrast, exhibit expression patterns with respect tothese genes that are different from transgenic mice known in the art.

Replacement of non-human genes in a non-human animal with homologous ororthologous human genes or human sequences, at the endogenous non-humanlocus and under control of endogenous promoters and/or regulatoryelements, can result in a non-human animal with qualities andcharacteristics that may be substantially different from a typicalknockout-plus-transgene animal. In the typical knockout-plus-transgeneanimal, an endogenous locus is removed or damaged and a fully humantransgene is inserted into the animal's genome and presumably integratesat random into the genome. Typically, the location of the integratedtransgene is unknown; expression of the human protein is measured bytranscription of the human gene and/or protein assay and/or functionalassay. Inclusion in the human transgene of upstream and/or downstreamhuman sequences are apparently presumed to be sufficient to providesuitable support for expression and/or regulation of the transgene nomatter where in the animal's genome the transgene winds up. But in manycases the transgene with human regulatory elements expresses in a mannerthat is unphysiological or otherwise unsatisfactory, and can be actuallydetrimental to the animal. In contrast, the inventors demonstrate that areplacement with human sequence at an endogenous locus under control ofendogenous regulatory elements provides a physiologically appropriateexpression pattern and level that results in a useful humanized animalwhose physiology with respect to the replaced gene are meaningful andappropriate and context of the humanized animal's physiology.

Fertilized mouse eggs injected with a construct having the MHC class Ipromoter H2 and a β-globin intron driving expression of a 695-bp mouseIL-6 gene reportedly produce mice that constitutively express mouse IL-6at relatively high levels (as compared with wild-type mice) (see,Woodrofe et al. (1992) Long-Term Consequences of Interleukin-6Overexpression in Transgenic Mice, DNA and Cell Biology 11(8):587-592).But these mice are prone to develop lymphomas associated with theintestines, lymph nodes, and kidney, as well as splenic amyloiddeposits. They also exhibit abnormal B cell maturation (see, Woodrofe etal., Id.), so that studies of B cell function are compromised. Incontrast, mice as described herein that comprise a replacement of themouse IL-6 gene with a human IL-6 gene at the mouse IL-6 locus are notprone to develop these lymphomas, and the mice exhibit apparently normalB cell populations.

Mice (C57BL/6) transgenic for hIL-6 due to a random insertion of a6.6-kb (BamHI-Pvu II fragment) length of human DNA containing the hIL-6gene coupled with an IgM enhancer have been reported (see, Suematsu etal. (1989) IgG1 plasmocytosis in interleukin 6 transgenic mice, Proc.Natl. Acad. Sci. USA 86:7547-7551). The mice express hIL-6 at between800 pg/mL and 20,000 pg/mL in serum, where wild-type mice typicallyexpress only about 100 pg/mL IL-6. The mice exhibit an increase in serumIg (120 to 400-fold over wild-type mice) and a decrease in albumin asthey age. The mice suffer from a massive plasmacytosis, exhibitsplenomegaly and lymph node enlargement, as well as exhibiting plasmacells and increased megakaryocytes in bone marrow. Upon inspection, whatappear to be enlarged lymph nodes are instead massed of compactedabnormal plasma cells. Both spleen and thymus exhibit massiveproliferation of plasma cells, which also infiltrate portions of thelung, liver, and kidney. Kidney in these mice also exhibitsIL-6-stimulated mesangial cell proliferation typical ofmesangio-proliferative glomerulonephritis. Similarly, mice (BALB/c)transgenic for a trimmed hIL-6 cDNA driven by a mouse H-2L^(d) promoterrandomly inserted into the genome display severe plasmacytosis (see,Suematsu et al. (1992) Generation of plasmacytomas with the chromosomaltranslocation t(12;15) in interleukin 6 transgenic mice, Proc. Natl.Acad. Sci. USA 89:232-235). Although C57BL/6 mice that overexpress hIL-6do not develop transplantable plasmacytomas (they do exhibitplasmacytosis), transgenic BL/6 mice back-crossed into BALB/c micereportedly do.

Random transgenesis of a hIL-6 cDNA driven by a glial fibrillary acidicprotein (GFAP) gene promoter reportedly results in hIL-6 overexpressionin the mouse central nervous system, which also leads to significantpathologies (see, Campbell et al. (1993) Neurologic disease induced intransgenic mice by cerebral overexpression of interleukin 6, Proc. Natl.Acad. Sci. USA 90:10061-10065). These mice exhibit extensiveneuropathology and reactive astrocytosis resulting from IL-6 expressionin the CNS due to loss of control as the result of random integration ofan IL-6 transgene at an apparently CNS-permissive transcriptional locus.Although expression of hIL-6 cDNA linked to a β-globin 3′-UTR and drivenby a neuron-specific enolase promoter microinjected into fertilizedmouse eggs (F1 C57BL/6× BALB/c) produced mice with a normal lifespan andwithout apparent neurological defects that expressed hIL-6 in neuronsbut not elsewhere (see, Fattor et al. (1994) IL-6 Expression in Neuronsof Transgenic Mice Causes Reactive Astrocytosis and Increase in RamifiedMicroglial Cells But No Neuronal Damage, Eur. J. Neuroscience7:2441-2449), the mice exhibited high levels (20- to 30-fold higher thanwild-type) of activated and enlarged astrocytes with increased processesthroughout the brain, as well as a 10- to 15-fold increase in ramifiedmicroglial cells in white matter. Thus, brain expression of IL-6reportedly leads to conditions that range from reactive astrocytosis tofrank and profound neuropathology.

Microinjection into fertilized eggs of an F1 cross of C57BL/6×“DBAII”mice of a 639-bp hIL-6 cDNA linked to a β-globin 3′-UTR and a mouse MT-1promoter reportedly produced a transgenic mouse in which the hIL-6 genewas randomly integrated produced a weakened and diseased mouse that diesyoung of kidney failure (see Fattori et al. (1994) Blood, Development ofProgressive Kidney Damage and Myeloma Kidney in Interleukin-6 TransgenicMice, Blood 63(9):2570-2579). Transgenic mice expired at 12-20 weeks andexhibited elevated levels of al and β-globulins in plasma,hypergammaglobulinemia, elevated megakaryocytes in spleen (3-fold higherthan wild-type) and bone marrow, plasmacytosis of lymphoid organs(spleen, thymus, and lymph nodes) characterized by abnormal andcompactly arranged plasmocytoid cells, and glomerulonephritis leading toglomerulosclerosis similar to multiple myeloma.

Microinjection into fertilized eggs of a C57BL/6J mouse of aH-2L^(d)-driven hIL-6 cDNA caused IL-6-dependent muscle wasting in mice,characterized in part by a significantly lower gastrocnemius muscleweight in transgenic mice as compared to weight-matched controls, adifference that was ameliorated by treatment with an IL-6 antagonist(see, Tsujinaka et al. (1996) Interleukin 6 Receptor Antibody InhibitsMuscle Atrophy and Modulates Proteolytic Systems in Interleukin 6Transgenic Mice, J. Clin. Invest. 97(1):244-249). At 12 weeks these micedisplayed serum hIL-6 levels of more than 600,000 pg/mL. The transgenicmice also had livers that weighed about 1,242 mg, as compared to controllivers that were about 862 mg. Transgenic mice treated with IL-6antagonist had livers that weighed about 888 mg. Muscle cathepsins B andB+L were significantly higher (20-fold and 6.2-fold) in transgenic micethan in controls, a phenomenon that was eliminated in transgenic micetreated with an IL-6 antagonist. cathepsin B and L mRNAs were estimatedto be about 277% and 257%, respectively, as compared with wild-typemice; the difference was significantly reduced with IL-6 antagonisttreatment.

Mice comprising a hIL-6 minigene driven by a mouse MHC class I H-2Ldpromoter and a hIL-6R minigene driven by a chicken β-actin promoter, anda gp130 gene, exhibited pathologies typical of hIL-6 transgenic mice(e.g., hepergammaglobulinemia, splenomegaly, mesangial proliferativeglomerulonephritis, lung lymphoid infiltration) as well as ventricularhypertrophy (Hirota et al. (1995) Continuous activation of gp130, asignal-transducing receptor component for interleukin 6-relatedcytokines, causes myocardial hypertrophy in mice, Proc. Natl Acad. Sci.USA 92:4862-4866). The ventricular hypertrophy is believed to bemediated by a continuous activation of gp130 (Id.). The role of IL-6 isreportedly to help strengthen the cytokine receptor complex and inducedimerization of gp130, which is the signal transducing componentresponsible for transducing the IL-6 signal (Paonessa et al. (1995) Twodistinct and independent sites on IL-6 trigger gp130 dimer formation andsignalling, EMBO J. 14(9):1942-1951). The activated complex is believedto be a hexamer composed of two IL-6, each IL-6 bound to one IL-6Rα andtwo gp130 (each IL-6 contains two independent gp130-binding sites)exhibiting a 2:2:2 stoichiometry, wherein the dimerization of gp130causes activation of JAK-Tyk tyrosine kinases, phosphorylation of gp130and STAT family transcription factors and other intracellular substrates(Id.; Stahl, N. (1994) Association and Activation of Jak-Tyk Kinases byCNTF-LIF-OSM-IL-6 l Receptor Components, Science 263:92-95), consistentwith a general model of cytokine receptor complex formation (see, Stahl,N. and Yancopoulos, G. (1993) The Alphas, Betas, and Kinases of CytokineReceptor Complexes, Cell 74:587-590; Davis et al. (1993) LIFIRβ andgp130 as Heterodimerizing Signal Transducers of the Tripartite CNTFReceptor, Science 260:1805-1808; Murakami et al. (1993) IL-6-InducedHomodimerization of gp130 and Associated Activation of a TyrosineKinase, Science 2601808-1810).

Mice transgenic for human sIL-6R driven by a rat PEP carboxykinasepromoter and human IL-6 driven by a mouse metallothionein-1 promoter arereportedly markedly smaller that mice transgenic for human IL-6 alone orhuman sIL-6R alone (Peters et al. (1997) Extramedullary Expansion ofHematopoietic Progenitor Cells in Interleukin(IL-)-6-sIL-6R DoubleTransgenic Mice, J. Exp. Med. 185(4):755-766), reflected in reduced bodyfat and reduced weight (20-25 g vs. 40 g). Double transgenic micereportedly also exhibit spleen (5-fold) and liver (2-fold) enlargementas compared with reportedly normal organ weights for single transgenicmice, apparently due to extramedullary proliferation of hematopoeiticcells of spleen and liver but not bone marrow, as well as elevatedmegakaryocytes in spleen and plasmacellular infiltrates in allparenchymal organs (Id.). Double transgenics also exhibit livers with anincrease of about 200- to about 300-fold in granulocytes, macrophages,progenitor cells, and B cells as compared with single transgenics; incontrast, IL-6 single transgenic mice exhibited lesser increases inmacrophages (15-fold) and B cells (45-fold) (Id.). The extraordinaryfindings are presumably due to stimulation of growth and differentiationof hematopoietic progenitor cells by activating gp130 signaltransduction (Id.).

Further, double transgenic (mouse metallothionine promoter-drivenhIL-6/rat PEP carboxykinase promoter-driven hIL-6R) mice exhibit ahepatocellular hyperplasia that is reportedly identical to human nodularregenerative hyperplasia with sustained hepatocyte proliferation thatstrongly suggests that IL-6 is responsible for both hepatocyteproliferation and pathogenic hepatocellular transformation (Maione etal. (1998) Coexpression of IL-6 and soluble IL-6R causes nodularregenerative hyperplasia and adenomas of the liver, EMBO J.17(19):5588-5597). Because hepatocellular hyperplasia is reportedly notobserved in single transgenic hIL-6 mice and hIL-6 can bind mIL-6R, thefinding may appear paradoxical until it is considered that the doubletransgenic may result in higher levels of hIL-6 complexed to solubleIL-6R (here, soluble hIL-6R), which complex is a more potent inhibitorthat IL-6 alone (Id.).

In contrast to mice that are transgenic for human IL-6, humanized IL-6mice that comprise a replacement at an endogenous mouse IL-6 locus,which retain mouse regulatory elements but comprise a humanization ofIL-6-encoding sequence, do not exhibit the severe pathologies of priorart mice. Genetically modified mice that were heterozygous or homozygousfor hIL-6 were grossly normal.

Mice with a humanized IL-6 gene (MAID 760) as described in the Exampleswere immunophenotyped and found to have normal B cell numbers in FACSanalyses (lymphocyte-gated) of spleen B cells using a pan B cell marker(CD445R(B220)) (FIG. 4). For spleen, wild-type mice exhibited 63% Bcells; hIL-6 heterozygote mice exhibited 63% B cells; and micehomozygous for hIL-6 at the endogeous mouse locus exhibited 63% B cells.B cell numbers for homozygous hIL-6 mice immunized with TNP-KLH werealso normal (65% for wild-type, and 61% for hIL-6 homozygotes).

Splenic T cells were also about the same as wild-type (FIG. 5).Percentages of splenic T cells for Thelper/Tcytoxic were, for wild-type20%/40% (ratio of 1.4:1); for hIL-6 heterozygotes 23%/14% (ratio of1.6:1); for hIL-6 homozygotes 21%/15% (ratio of 1.4:1) (markers wereCD8a-APC; CD4-FITC). Homozygous hIL-6 mice immunized with TNP-KLHexhibited similar splenic T cell numbers to wild-type mice, i.e.,Thelper/Tcytotoxic were 22%/20% (ratio of 1.1:1) as compared with21%/19% for wild-type (also a ratio of 1.1:1).

Humanized IL-6 mice also exhibited about normal levels of splenic NKcells on FACS analysis (CD11b and DX5) (FIG. 7). hIL-6 heterozygotesexhibited 2.2% NK cells, and hIL-6 homozygotes exhibited 1.8% NK cells,whereas wild-type mice exhibited 2.4% NK cells. Following immunizationwith TNP-KLH, homozygotes exhibited 1.6% splenic NK cells, whereaswild-type mice exhibited 2.1% splenic NK cells.

Humanized IL-6 mice also exhibited normal levels of splenic Ly6G/C(Gr1)cells (FIG. 6). hIL-6 heterozygotes exhibited 7.0% GR1⁺ cells (1.3%Gr1^(hi)); homozygotes exhibited 6.8% Gr1⁺ cells (0.9% Gr1^(hi)),whereas wild-type mice exhibited 8.0% Gr1⁺ cells (1.8% Gr1^(hi)).Immunized IL-6 homozygotes (immunized with TNP-KLH) exhibited 11% Gr1⁺cells (4.0% Gr1^(hi)), whereas wild-type mice exhibited 10% Gr1⁺ cells(3.0% Gr1^(hi)).

Humanized IL-6 mice also exhibited normal blood B and T cell numbers inFACS analysis (FIG. 8 and FIG. 9). FACs with a pan B cell marker(CD445R(B220)) revealed that homozygous hIL-6 mice exhibited 52% B cellas compared with wild-type 53%; heterozygotes exhibited 38% (an averageof two different stainings of 29% and 47%). Homozygous hIL-6 miceimmunized with TNP-KLH gave similar B cell numbers (43%, as comparedwith 45% for wild-type mice).

Humanized IL-6 mice exhibited normal blood T cell numbers in FACSanalysis as measured by CD8a and CD4 staining. Heterozygous hIL-6 miceexhibited Thelper/Tcytotoxic numbers of 39%/26% (ratio of 1.5:1);homozygous hIL-6 mice exhibited Th/Tc numbers of 24%/20% (ratio of1.2:1), whereas wild-type mice exhibited Th/Tc numbers of 26%/20% (ratioof 1.3:1). Homozygous hIL-6 mice immunized with TNP-KLH had Th/Tcnumbers of 29%/21% (ratio of 1.4:1), whereas wild-type immunized micehad Th/Tc numbers of 28%/23% (1.2:1).

Humanized IL-6 mice also exhibited myeloid cell numbers in blood thatwere similar to wild-type mice as measured by FACS analysis of naïve andimmunized mouse blood stained with Ly6G/C(Gr1) and CD11b, as well asCD11b and DX5 (FIG. 10, FIG. 11, and FIG. 12). Heterozygous hIL-6 miceexhibited % Gr+ cells of 10.8%, homozygotes 6.9%, whereas wild-type miceexhibited 9.7%. Immunized hIL-6 homozygotes exhibited M1(Ly6G/C(Gr) of10¹-10⁴)/M2(Ly6G/C(Gr) staining of about 10²-10³) numbers of 43%/34%,whereas wild-type mice had numbers of 45%/38%. FACS plots of CD11b(vertical axis) vs. Ly6G/C (horizontal axis) for immunized homozygoushIL-6 mice showed cell percentage in quadrants (upper left/upperright/lower right) of 16%/8%/3%, which were identical to immunizedwild-type quadrant numbers.

Homozygous TNP-KLH-immunized humanized IL-6 mice exhibited CD11b vs.DX5(NK) staining FACS plots that were similar to immunized wild-typemice. Quadrant analysis blood FACS plots (CD11b vertical axis, DX5(NK)horizontal axis) revealed upper left/upper right/lower right numbers of9.5%/17%/10% for hIL-6 homozygotes and 6.5%/17.3%/14% for wild-typemice.

Humanized IL-6 mice exhibited an isotype response that was essentiallythe same as observed in wild-type mice. Early and final IgG1, IgG2a,IgG2b, IgG3, IgA, IgE, and IgM levels were about the same as observed inwild-type mice. In one experiment, final IgM was slightly higher inhumanized mice; final IgG3 was also elevated in humanized mice.

B cell development in naïve hIL-6 mice was essentially indistinguishablefrom development in wild-type mice based on FACS analysis of bone marrowIgM/CD24/B220 staining (FIG. 13). Immunophenotyping of immune micerevealed that marker populations for various cell types in the B celldevelopment progression were essentially normal in hIL-6 mice.Progression of cells from hematopoietic stem cells, common lymphoidprogenitors, ProB cells, PreB cells, and immature and mature B cells isnormal in hIL-6 mice (FIG. 14 and FIG. 15)

EXAMPLES Example 1 Replacement of Endogenous Mouse IL-6 Gene with hIL-6Gene

The 4.8-kb human IL-6 gene containing exons 1 through 5 of the humanIL-6 gene replaced 6.8 kb of the murine IL-6 gene locus.

A targeting construct for replacing the mouse with the human IL-6 genein a single targeting step was constructed using VELOCIGENE® geneticengineering technology (see, Valenzuela et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech, 21(6):652-659). Mouse and human IL-6 DNA wereobtained from bacterial artificial chromosome (BAC) RPCI-23 clone 368C3,and from BAC CTD clone 2369M23, respectively. Briefly, a NotI linearizedtargeting construct generated by gap repair cloning containing mouseIL-6 upstream and downstream homology arms flanking a 4.8 kb human IL-6sequence extending from ATG in exon 1 to exon 5 with 16 nucleotides of3′ downstream sequence (genomic coordinates: NCBIh37.1: ch7:22,766,882to 22,771,637) and a floxed neo selection cassette, was electroporatedinto F1H4 mouse embryonic stem (ES) cells (C57BL/6×129 F1 hybrid).Correctly targeted ES cells (MAID 790) were further electroporated witha transient Cre-expressing vector to remove the drug selection cassette.Targeted ES cell clones without drug cassette (MAID 1428) wereintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, U.S. Pat. No. 7,294,754, 7,576,259, 7,659,442, and Poueymirou etal. (2007) F0 generation mice that are essentially fully derived fromthe donor gene-targeted ES cells allowing immediate phenotypic analysesNature Biotech. 25(1):91-99). VELOCIMICE® (F0 mice fully derived fromthe donor ES cell) bearing the humanized IL-6 gene were identified bygenotyping for loss of mouse allele and gain of human allele using amodification of allele assay (see, Valenzuela et al. (2003)).

Correctly targeted ES cell clones were identified by aloss-of-native-allele (LONA) assay (Valenzuela et al. 2003) in which thenumber of copies of the native, unmodified II6 gene were determined bytwo TaqMan™ quantitative polymerase chain reactions (qPCRs) specific forsequences in the mouse II6 gene that were targeted for deletion. TheqPCR assays comprised the following primer-probe sets (written 5′ to3′): upstream forward primer, TTGCCGGTTT TCCCTTTTCT C (SEQ ID NO:1);upstream reverse primer, AGGGAAGGCC GTGGTTGTC (SEQ ID NO:2); upstreamprobe, FAM-CCAGCATCAG TCCCAAGAAG GCAACT-BHQ (SEQ ID NO:3); downstreamforward primer, TCAGAGTGTG GGCGAACAAA G (SEQ ID NO:4); downstreamreverse primer, GTGGCAAAAG CAGCCTTAGC (SEQ ID NO:5); downstream probe,FAM-TCATTCCAGG CCCTTCTTAT TGCATCTG-BHQ (SEQ ID NO:6); in which FAMrefers to the 5-carboxyfluorescein fluorescent probe and BHQ refers tothe fluorescence quencher of the black hole quencher type (BiosearchTechnologies). DNA purified from ES cell clones that that have taken upthe targeting vector and incorporated in their genomes was combined withTaqMan™ Gene Expression Master Mix (Life Technologies) according to themanufacturer's suggestions in a 384-well PCR plate (MicroAmp™ Optical384-Well Reaction Plate, Life Technologies) and cycled in an AppliedBiosystems Prism 7900HT, which collects fluorescence data during thecourse of the PCRs and determines a threshold cycle (Ct), the fractionalPCR cycle at which the accumulated fluorescence reaches a pre-setthreshold. The upstream and downstream II6-specific qPCRs and two qPCRsfor non-targeted reference genes were run for each DNA sample. Thedifferences in the Ct values (ΔCt) between each II6-specific qPCR andeach reference gene qPCR were calculated, and then the differencebetween each ΔCt and the median ΔCt for all samples assayed wascalculated to obtain ΔΔCt values for each sample. The copy number of theII6 gene in each sample was calculated from the following formula: copynumber=2˜2^(−ΔΔCt). A correctly targeted clone, having lost one of itsnative copies, will has an II6 gene copy number equal to one.Confirmation that the human IL6 gene sequence replaced the deleted mouseII6 gene sequence in the humanized allele was confirmed by a TaqMan™qPCR assay that comprises the following primer-probe sets (written 5′ to3′): the human forward primer, CCCCACTCCACTGGAATTTG (SEQ ID NO:7); thehuman reverse primer, GTTCAACCACAGCCAGGAAAG (SEQ ID NO:8); and the humanprobe, FAM-AGCTACAACTCATTGGCATCCTGGCAA-BHQ (SEQ ID NO:9).

The same LONA assay was used to assay DNA purified from tail biopsiesfor mice derived from the targeted ES cells to determine their II6genotypes and confirm that the humanized II6 allele had transmittedthrough the germline. Two pups heterozygous for the replacement are bredto generate a mouse that is homozygous for the replacement of theendogenous mouse IL-6 gene by the human IL-6 gene. Pups that arehomozygous for the replacement are used for phenotyping.

The upstream junction of the murine locus and the sequence containingthe hIL-6 gene is designed to be within 5′-AATTAGAGAG TTGACTCCTAATAAATATGA GACTGGGGAT GTCTGTAGCT CATTCTGCTC TGGAGCCCAC CAAGAACGATAGTCAATTCC AGAAACCGCT ATGAACTCCT TCTCCACAAG TAAGTGCAGG AAATCCTTAGCCCTGGAACT GCCAGCGGCG GTCGAGCCCT GTGTGAGGGA GGGGTGTGTG GCCCAGG (SEQ IDNO:10), wherein the final mouse nucleotide prior to the first nucleotideof the human gene is the “T” in CCGCT, and the first nucleotide of thehuman sequence is the first “A” in ATGAA. The downstream junction of thesequence containing the hIL-6 gene and the murine locus is designed tobe within 5′-TTTTAAAGAA ATATTTATAT TGTATTTATA TAATGTATAA ATGGTTTTTATACCAATAAA TGGCATTTTA AAAAATTCAG CAACTTTGAG TGTGTCACGC TCCCGGGCTCGATAACTATA ACGGTCCTAA GGTAGCGACT CGAGATAACT T-3′ (SEQ ID NO:11), whereinthe final nucleotide of the human sequence is with the final “G” inTCACG and the first nucleotide of the mouse sequence is the first “C” inCTCCC; the downstream junction region also contained a loxP site at the3′ end (the beginning of which is shown) for removal of a floxedubiquitin promoter-driven neo cassette. The junction of the neo cassettewith the mouse IL-6 locus is designed to be within 5′-TATACGAAGTTATCCTAGGT TGGAGCTCCT AAGTTACATC CAAACATCCT CCCCCAAATC AATAATTAAGCACTTTTTAT GACATGTAAA GTTAAATAAG AAGTGAAAGC TGCAGATGGT GAGTGAGA (SEQ IDNO:12), where the final “C” of AGCTC is the final nucleotide of the neocassette; the first nucleotide of the mouse genome following thecassette is the initial “C” of CTAAG.

Example 2 Immunophenotyping of Naive and Immunized hIL-6 Mice: B Cells

Mice homozygous for the hIL-6 gene replacement were analyzed for B cells(DC445R(B220). Lymphocyte-gated fractions from spleen cell preparationsof naive and immunized (TNP-KLH) hIL-6 mice were stained andimmunophenotyped using flow cytometry. FACS analysis showed that thepercentage of B cells of the spleen cell preparation as measured byCD45R(B220)-FITC staining were about the same (63% of cells) forpreparations from naive wild-type, hIL-6 heterozygotes, and hIL-6homozygotes. For immunized mice, B cells accounted for about 65% oftotal cells of the spleen cell preparation in wild-type mice, and about61% of total cells in hIL-6 homozygotes. Spleens of hIL-6 mice (bothnaive and immunized) contain a population of B cells that is about thesame size as the splenic B cell population in wild-type mice.

Bone marrow of wild-type, hIL-6 heterozygotes, and hIL-6 homozygotes wasstained with B cell markers (CD45R(B220)-APC, CD24(HSA)-PE, or CD43conjugated to a dye and/or IgM (IgM-FITC). B cell development in bonemarrow of normal mice will be reflected in surface markers as cellsprogress from stem cells to early pro-B cells to late pro-B cells, tolarge pre-B cells to small pre-B cells to immature B cells and finally,to mature B cells. Common lymphocyte progenitor pro-B cells will expressCD45R, and in later stages will express IgM as immature and later asmature B cells. Thus, CD45R-stained and anti-IgM-stained B cells shouldreveal a pattern characteristic of B cell development. Bone marrow ofhIL-6 heterozygotes and homozygotes displayed a pattern ofCD45R(B220)-APC and anti-IgM-FITC staining that was essentiallyindistinguishable from wild-type bone marrow, showing populations of Bcells that stained positive for CD45R(B220) and IgM, or CD45R(B220)alone. B cell sub-populations in bone marrow of hIL-6 mice revealed byFACS staining were similar to those in wild-type mice (Table 1; seealso, FIG. 13).

TABLE 1 B Cells in Bone Marrow of Naive Mice hIL-6 Mouse Wild-type MouseHeterozygote Homozygote (%) (%) (%) CLP-ProB 40 29 32 PreB-ImmatureB12.3 19.3 15.3 Mature B 6.4 6.5 6.7

Staining for CD24 (see FIG. 14) revealed the (normal) pattern shown inTable 2, indicating normal development in bone marrow.

TABLE 2 B Cells in Bone Marrow of Naive Mice hIL-6 Mouse Wild-type MouseHeterozygote Homozygote (%) (%) (%) Developing HSC- 46.6 46 43 CLPMature CLP/early 10.2 9.0 10.1 ProB Late ProB, PreB, 7.2 11.6 10.7Immature B Mature B 14.1 14.9 17

Staining for CD43 (see FIG. 15) revealed the (normal) pattern shown inTable 3, indicating normal development in bone marrow.

TABLE 3 B Cells in Bone Marrow of Naive Mice hIL-6 Mouse Wild-type MouseHeterozygote Homozygote (%) (%) (%) PreBII-Immature 28.4 21.4 21.2 Bcells Mature B cells 8.1 11.5 8.0 ProB-PreBI 3.4 4.3 4.7

Thus, immunophenotyping of naïve hIL-6 mice revealed that B celldevelopment in such mice is essentially normal.

Example 3 Replacement of Endogenous Mouse IL-6Rα Ectodomain GeneSequence with hIL-6Rα Ectodomain Gene Sequence

The 45 kb human IL-6Rα gene containing exons 1 through 8 of the humanIL-6Rα gene replaced 35.4 kb of the murine IL-6Rα gene locus. Mouseexons 9 and 10 were retained; only exons 1-8 were humanized. In total,35,384 nt of mouse sequence was replaced by 45,047 nt of human sequence.

A targeting construct for replacing the mouse with the human IL-6Rα genein a single targeting step was constructed using VELOCIGENE® geneticengineering technology (see, Valenzuela et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech, 21(6):652-659). Mouse and human IL-6Rα DNAwere obtained from bacterial artificial chromosome (BAC) RPCI-23 clone125J8, and from BAC CTD clone 2192J23, respectively. Briefly, a NotIlinearized targeting construct generated by gap repair cloningcontaining mouse IL-6Rα upstream and downstream homology arms flanking a45 kb human IL-6Rα sequence extending from ATG in exon 1 to exon 8 with69 nucleotides of 3′ downstream sequence and a floxed neo selectioncassette, was electroporated into F1H4 mouse embryonic stem (ES) cells(C57BL/6×129 F1 hybrid). Correctly targeted ES cells (MAID 794) werefurther electroporated with a transient Cre-expressing vector to removethe drug selection cassette. Targeted ES cell clones without drugcassette (MAID 1442) were introduced into an 8-cell stage mouse embryoby the VELOCIMOUSE® method (see, U.S. Pat. Nos. 7,294,754, 7,576,259,7,659,442, and Poueymirou et al. (2007) F0 generation mice that areessentially fully derived from the donor gene-targeted ES cells allowingimmediate phenotypic analyses Nature Biotech. 25(1):91-99). VELOCIMICE®(F0 mice fully derived from the donor ES cell) bearing the humanizedIL-6Rα gene were identified by genotyping for loss of mouse allele andgain of human allele using a modification of allele assay (see,Valenzuela et al. (2003)).

Correctly targeted ES cell clones were identified by aloss-of-native-allele (LONA) assay (Valenzuela et al. 2003) in which thenumber of copies of the native, unmodified II6 gene were determined bytwo TaqMan™ quantitative polymerase chain reactions (qPCRs) specific forsequences in the mouse II6 gene that were targeted for deletion. TheqPCR assays comprised the following primer-probe sets (written 5′ to3′): upstream forward primer, GCCCTAGCAT GCAGAATGC (SEQ ID NO:13);upstream reverse primer, AAGAGGTCCC ACATCCTTTG C (SEQ ID NO:14);upstream probe, CCCACATCCA TCCCAATCCT GTGAG (SEQ ID NO:15); downstreamforward primer, GAGCTTGCCC CCAGAAAGG (SEQ ID NO:16); downstream reverseprimer, CGGCCACATC TCTGGAAGAC (SEQ ID NO:17); downstream probe,CATGCACTGC CCCAAGTCTG GTTTCAGT (SEQ ID NO:18). DNA purified from ES cellclones that that have taken up the targeting vector and incorporated intheir genomes was combined with TaqMan™ Gene Expression Master Mix (LifeTechnologies) according to the manufacturer's suggestions in a 384-wellPCR plate (MicroAmp™ Optical 384-Well Reaction Plate, Life Technologies)and cycled in an Applied Biosystems Prism 7900HT, which collectsfluorescence data during the course of the PCRs and determines athreshold cycle (Ct), the fractional PCR cycle at which the accumulatedfluorescence reaches a pre-set threshold. The upstream and downstreamIL-6Rα-specific qPCRs and two qPCRs for non-targeted reference geneswere run for each DNA sample. The differences in the Ct values (ΔCt)between each IL-6Rα-specific qPCR and each reference gene qPCR werecalculated, and then the difference between each ΔCt and the median ΔCtfor all samples assayed was calculated to obtain ΔΔCt values for eachsample. The copy number of the II6 gene in each sample was calculatedfrom the following formula: copy number=2·2-ΔΔCt. A correctly targetedclone, having lost one of its native copies, will have an IL-6Rα genecopy number equal to one. Confirmation that the human IL-6Rα genesequence replaced the deleted mouse IL-6Rα gene sequence in thehumanized allele was confirmed by a TaqMan™ qPCR assay that comprisesthe following primer-probe sets (written 5′ to 3′): the human forwardprimer, GGAGAGGGCA GAGGCACTTA C (SEQ ID NO:19); the human reverseprimer, GGCCAGAGCC CAAGAAAAG (SEQ ID NO:20); and the human probe,CCCGTTGACT GTAATCTGCC CCTGG (SEQ ID NO:21).

The same LONA assay was used to assay DNA purified from tail biopsiesfor mice derived from the targeted ES cells to determine their IL-6Rαgenotypes and confirm that the humanized IL-6Rα allele had transmittedthrough the germline. Pups heterozygous for the replacement are bred togenerate a mouse that is homozygous for the replacement of theendogenous mouse IL-6Rα gene by the human IL-6Rα (ectodomain) gene. Pupsthat are homozygous for the replacement are used for phenotyping.

The upstream junction of the murine locus and the sequence containingthe hIL-6Rα gene is designed to be within 5′-CGAGGGCGAC TGCTCTCGCTGCCCCAGTCT GCCGGCCGCC CGGCCCCGGC TGCGGAGCCG CTCTGCCGCC CGCCGTCCCGCGTAGAAGGA AGCATGCTGG CCGTCGGCTG CGCGCTGCTG GCTGCCCTGC TGGCCGCGCCGGGAGCGGCG CTGGCCCCAA GGCGCTGCCC TGCGCAGGGT AAGGGCTTCG G (SEQ ID NO:22),wherein the final mouse nucleotide prior to the first nucleotide of thehuman gene is the “C” in GAAGC, and the first nucleotide of the humansequence is the first “A” in ATGCT. The downstream junction of thesequence containing the hIL-6 gene and the murine locus is designed tobe within 5′-CAAGATTATT GGAGTCTGAA ATGGAATACC TGTTGAGGGA AATCTTTATTTTGGGAGCCC TTGATTTCAA TGCTTTTGAT TCCCTATCCC TGCAAGACCC GGGCTCGATAACTATAACGG TCCTAAGGTA GCGACTCGAG ATAACTTC-3′ (SEQ ID NO:23), wherein thefinal nucleotide of the human sequence is with the final “A” in CAAGAand the first nucleotide of the mouse sequence is the first “C” inCCCGG; the downstream junction region also contained a loxP site at the3′ end for removal of a floxed ubiquitin promoter-driven neo cassette.The first nucleotide of the loxp site is the first “A” in ATAAC. Thejunction of the neo cassette with the mouse IL-6Rα locus is designed tobe within 5′-TATACGAAGT TATCCTAGGT TGGAGCTCTA CTCCATATGC TCACTTGCCGTTGTTTGCTA CGATACGGTG AGGCCCGTGC GAAGAGTGGC ACAGATCAGG AGGCTTATGTGGTCAGTCCA CAGTATGGC (SEQ ID NO:24), where the final “C” of AGCTC is thefinal nucleotide of the neo cassette; the first nucleotide of the mousegenome following the cassette is the initial “T” of TACTC.

We claim:
 1. A genetically modified mouse, wherein the genome of saidmouse comprises a replacement at an endogenous mouse IL-6 locus of amouse gene encoding IL-6 with a human gene encoding human IL-6, whereinthe human gene encoding human IL-6 is under control of endogenous mouseregulatory elements at the endogenous mouse IL-6 locus and is expressed,and the replaced mouse IL-6 gene is not expressed; wherein the genome ofsaid mouse further comprises a replacement at an endogenous mouse IL-6Rαlocus of a mouse nucleic acid encoding a mouse IL-6Rα ectodomain with ahuman nucleic acid encoding a human IL-6Rα ectodomain to form ahumanized IL-6Rα gene, wherein the humanized IL-6Rα gene is undercontrol of endogenous mouse regulatory elements at the endogenous mouseIL-6Rα locus, wherein the humanized IL-6Rα gene is expressed and encodesa humanized IL-6Rα protein which comprises said human IL-6Rα ectodomainand mouse IL-6Rα transmembrane and cytoplasmic domains; and wherein themouse exhibits an acute phase response to an acute phase inducer.
 2. Thegenetically modified mouse of claim 1, wherein the mouse does notexhibit a feature selected from plasmocytosis, glomerulosclerosis,glomerulonephritis, kidney failure, hypergammaglobulinemia, elevatedmegakaryocytes in spleen, elevated megakaryocytes in bone marrow,splenomegaly, lymph node enlargement, compacted abnormal plasma cells,and a combination thereof.
 3. A method for making a geneticallymodified, humanized mouse, comprising: i) replacing an endogenous mousegene sequence encoding mouse IL-6 at an endogenous mouse IL-6 locus witha human gene encoding human IL-6, and ii) replacing a nucleic acidencoding the mouse IL-6Rα ectodomain at an endogenous mouse IL-6Rα locuswith a nucleic acid encoding the human IL-6Rα ectodomain to form ahumanized IL-6Rα gene, to make a genetically modified, humanized mousewhose genome comprises a replacement of the endogenous mouse geneencoding IL-6 and a replacement of the nucleic acid encoding theendogenous mouse IL-6Rα ectodomain with corresponding human sequences,wherein the human gene encoding human IL-6 is operably linked toendogenous mouse IL-6 regulatory sequences such that the gene encodinghuman IL-6 is expressed and the replaced mouse IL-6 gene is notexpressed, and wherein the humanized IL-6Rα gene is operably linked toendogenous mouse IL-6Rα regulatory sequences such that the humanizedIL-6Rα gene is expressed and encodes a humanized IL-6Rα protein thatcomprises said human IL-6Rα ectodomain and mouse IL-6Rα transmembranedomain and cytoplasmic domain, and wherein the mouse exhibits an acutephase response to an acute phase inducer.
 4. The genetically modifiedmouse of claim 1, wherein said mouse is heterozygous with respect to thereplacement at the endogenous mouse IL-6 locus.
 5. The geneticallymodified mouse of claim 1, wherein said mouse is homozygous with respectto the replacement at the endogenous mouse IL-6 locus.
 6. Thegenetically modified mouse of claim 1, wherein said mouse isheterozygous with respect to said humanized IL-6Rα gene.
 7. Thegenetically modified mouse of claim 1, wherein said mouse is homozygouswith respect to said humanized IL-6Rα gene.
 8. The genetically modifiedmouse of claim 1, wherein said mouse is homozygous with respect to thereplacement at the endogenous mouse IL-6 locus, and wherein said mouseis homozygous with respect to said humanized IL-6Rα gene.
 9. Thegenetically modified mouse of claim 1, wherein said mouse does notexpress a mouse IL-6Rα.
 10. The genetically modified mouse of claim 1,wherein said human gene encoding human IL-6 comprises exon 1 throughexon 5 of a human IL-6 gene.
 11. The method of claim 3, wherein saidhumanized mouse is heterozygous with respect to the replacement at theendogenous mouse IL-6 locus.
 12. The method of claim 3, wherein saidhumanized mouse is homozygous with respect to the replacement at theendogenous mouse IL-6 locus.
 13. The method of claim 3, wherein saidhumanized mouse is heterozygous with respect to said humanized IL-6Rαgene.
 14. The method of claim 3, wherein said humanized mouse ishomozygous with respect to said humanized IL-6Rα gene.
 15. The method ofclaim 3, wherein said humanized mouse is homozygous with respect to thereplacement at the endogenous mouse IL-6 locus, and wherein said mouseis homozygous with respect to said humanized IL-6Rα gene.
 16. The methodof claim 3, wherein said humanized mouse does not express a mouseIL-6Rα.
 17. The method of claim 3, wherein said human gene encodinghuman IL-6 comprises exon 1 through exon 5 of a human IL-6 gene.